Method of use of quinolone compounds against pneumococcal and haemophilus bacteria

ABSTRACT

This invention relates, in part, to newly identified methods of using quinolone antibiotics, particularly a gemifloxacin compound against certain pathogenic bacteria, particularly quinolone resistant S. pnemoniae and rare H. influenzae strains.

This application is a Continuation-in-Part to U.S. application Ser. No.09/399,657, filed Sep. 21, 1999, now abandoned U.S. ProvisionalApplication Nos. 60/141,456, filed Jun. 29, 1999, 60/142,749 filed Jul.8, 1999 and 60/142,725, filed Jul. 8, 1999. This invention relates, inpart, to newly identified methods of using quinolone antibiotics,particularly a gemifloxacin compound against pneumococcal andHaemophilus influenzae bacteria, such as Streptococcus pneumoniae,particularly quinolone-resistant strains, and Haemophilus strains,particularly rare strains of Haemophilus influenzae.

BACKGROUND OF THE INVENTION

Quinolones have been shown to be effective to varying degrees against arange of bacterial pathogens. However, as diseases caused by thesepathogens are on the rise, there exists a need for antimicrobialcompounds that are more potent than the present group of quinolones.

Gemifloxacin mesylate (SB-265805) is a novel fluoroquinolone useful as apotent antibacterial agent. Gemifloxacin compounds are described indetail in patent application PCT/KR98/00051 published as WO 98/42705.Patent application EP 688772 discloses novelquinolone(naphthyridine)carboxylic acid derivatives, including anhydrous(R,S)-7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylicacid of formula I.

PCT/KR98/00051 discloses(R,S)-7-(3-aminomethyl-4-syn-methoxyimino-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylicacid methanesulfonate and hydrates thereof including the sesquihydrate.

I. Pneumococcal Pathogens

The incidence of pneumococci resistant to penicillin G and otherβ-lactam and non-β-lactam compounds has increased worldwide at analarming rate, including in the U.S. Major foci of infections currentlyinclude South Africa, Spain, Central and Eastern Europe, and parts ofAsia (P. C. Appelbaum, Clin. Infect. Dis. 15:77-83, 1992; Friedland, etal., Pediatr. Infect. Dis. 11:433-435, 1992; Friedland, et al., N. Engl.J. Med. 331:377-382, 1994; Jacobs, et al., Clin. Infect. Dis.15:119-127, 1992 and Jacobs, et al., Rev. Med. Microbiol. 6:77-93,1995). In the U.S. a recent survey has shown an increase in resistanceto penicillin from <5% before 1989 (including <0.02% of isolates withMICs ≧2.0 μg/ml) to 6.6% in 1991-1992 (with 1.3% of isolates with MICs≧2.0 μg/ml) (Brieman, et al., J. Am. Med. Assoc. 271:1831-1835, 1994).In another more recent survey, 23.6% (360) of 1527 clinicallysignificant pneumococcal isolates were not susceptible to penicillin(Doern, et al., Antimicrob. Agents Chemother. 40:1208-1213, 1996). It isalso important to note the high rates of isolation of penicillinintermediate and resistant pneumococci (approximately 30%) in middle earfluids from patients with refractory otitis media, compared to otherisolation sites (Block, et al., Pediatr. Infect. Dis. 14:751-759, 1995).The problem of drug-resistant pneumococci is compounded by the abilityof resistant clones to spread from country to country, and fromcontinent to continent (McDougal, et al., Antimicrob. Agents Chemother.36:2176-2184, 1992; Munoz, et al., Clin. Infect. Dis. 15:112-118, 1992).

There is an urgent need of oral compounds for out-patient treatment ofotitis media and respiratory tract infections caused by penicillinintermediate and resistant pneumococci (Friedland, et al., Pediatr.Infect. Dis. 11:433-435, 1992; Friedland, et al., N. Engl. J. Med.331:377-382, 1994; M. R. Jacobs, Clin. Infect. Dis. 15:119-127, 1992;and Jacobs, et al., Rev. Med. Microbiol. 6:77-93, 1995). Availablequinolones such as ciprofloxacin and ofloxacin yield moderate in vitroactivity against pneumococci, with MICs clustering around thebreakpoints (Spangler, et al., Antimicrob. Agents Chemother. 36:856-859,1992; and Spangler, et al., J. Antimicrob. Chemother. 31:273-280, 1993).Gemifloxacin (SB 265805)(LB 20304a) is a new broad-spectrumfluoronaphthyridone carboxylic acid with a novel pyrrolidone substituent(Cormican, et al., Antimicrob. Agents Chemother. 41:204-211, 1997; Hohl,et al., Clin. Microbiol. Infect. 4:280-284, 1998; and Oh, et al.,Antimicrob. Agents Chemother. 40:1564-1568, 1996). Previous preliminarystudies (Cormican, et al., Antimicrob. Agents Chemother. 41:204-211,1997; Hohl, et al., Clin. Microbiol. Infect. 4:280-284, 1998; and Oh, etal., Antimicrob. Agents Chemother. 40:1564-1568, 1996) have shown thatthis compound is very active against pneumococci. This study furtherexamined the antipneumococcal activity of gemifloxacin compared tociprofloxacin, levofloxacin, sparfloxacin, grepafloxacin, trovafloxacin,amoxicillin, cefuroxime, azithromycin and clarithromycin by i) agardilution testing of 234 quinolone susceptible and resistant strains; ii)examination of resistance mechanisms in quinolone resistant strains;iii) time-kill testing of 12 strains; iv) examination of thepost-antibiotic effect (herein “PAE”) of drugs against 6 strains.

Provided herein is a significant discovery made using a gemifloxacincompound against a range of penicillin susceptible and resistantpneumococci by agar dilution, microdilution, time-kill andpost-antibiotic effect methodology. Against 64 penicillin susceptible,68 intermediate and 75 resistant pneumococci (all quinolonesusceptible), agar dilution MIC_(50/90) values (μg/ml) were as follows:gemifloxacin, 0.03/0.06; ciprofloxacin, 1.0/4.0; levofloxacin, 1.0/2.0;sparfloxacin, 0.5/0.5; grepafloxacin, 0.125/0.5; trovafloxacin,0.125/0.25; amoxicillin, 0.016/0.06 (penicillin susceptible), 0.125/1.0(penicillin intermediate), 2.0/4.0 (penicillin resistant); cefuroxime,0.03/0.25 (penicillin susceptible), 0.5/2.0 (penicillin intermediate),8.0/16.0 (penicillin resistant); azithromycin, 0.125/0.5 (penicillinsusceptible), 0.125/>128.0 (penicillin intermediate), 4.0/>128.0(penicillin resistant); clarithromycin, 0.03/0.06 (penicillinsusceptible), 0.03/32.0 (penicillin intermediate), 2.0/>128.0(penicillin resistant). Against 28 strains with ciprofloxacin MICs ≧8μg/ml, gemifloxacin had the lowest MICs (0.03-1.0 μg/ml, MIC₉₀ 0.5μg/ml), compared with MICs ranging between 0.25 to >32.0 μg/ml)(MIC₉₀s4.0→32.0 μg/ml) for the other quinolones. Resistance in these 28 strainswas associated with mutations in parC, gyrA, parE, and/or gyrb orefflux, with some strains having multiple resistance mechanisms. For 12penicillin susceptible and resistant pneumococcal strains (2 quinoloneresistant), time-kill results showed that levofloxacin at the MIC,gemifloxacin and sparfloxacin at 2×MIC and ciprofloxacin, grepafloxacinand trovafloxacin at 4×MIC, were bactericidal after 24 h. Gemifloxacinwas uniformly bactericidal after 24 h at ≦0.5 μg/ml. Various degrees of90% and 99% killing by all quinolones was detected after 3 h.Gemifloxacin and trovafloxacin were both bactericidal at the microbrothMIC for the two quinolone resistant pneumococcal strains. Amoxicillin,at 2×MIC and cefuroxime at 4×MIC, were bactericidal after 24 h, withsome degree of killing at earlier time periods. By contrast, macrolidesgave slower killing against the 7 susceptible strains tested, with 99.9%killing of all strains at 2-4×MIC after 24 h. Post-antibiotic effectsfor 5 quinolone susceptible strains were similar (0.3-3.0 h) for allquinolones tested, and significant quinolone PAEs were found for thequinolone-resistant strain.

Also provided herein is a significant discovery made using agemifloxacin compound against quinolone-resistant pneumococci,demonstrating the activity of the gemifloxacin compound used wassuperior to a number of quinolones as described in more detail herein.Gemifloxacin compounds are valuable compounds for the treatment ofinfections caused by a range of pneumococcal pathogens, including thoseresistant to usual oral therapy, thereby filling an unmet medical need.

II. Haemophilus Pathogens

Although development of an effective vaccine against Haemophilusinfluenzae type b has led to disappearance of this organism in manyparts of the world, its place has been taken by untypeable H. influenzaestrains. The latter organisms (followed by Streptococcus pneumoniae andMoraxella catarrhalis) are now considered to be the leading cause ofacute exacerbations of chronic bronchitis, and an important cause,together with S. pneumoniae and M. catarrhalis, of acute otitis media,sinusitis and conmmunity-acquired respiratory tract infections (Fang, etal., Medicine (Baltimore) 69:307-316, 1990; Hoberman, et al., Pediatr.Infect. Dis. 10:955-962, 1996; Jacobs, et al., Antimicrob. AgentsChemother, In press; and Zeckel, et al., Clin. Ther. 14:214-229, 1992).

Current recommendations by the NCCLS for use of Haemophilus Test Medium(herein “HTM”) for Haemophilus susceptibility testing have beencomplicated by difficulty in commercial manufacture of this medium, andits short balf-life when made in-house. Reliable Haemophilussusceptibility testing with HEM requires use of freshly made medium usedwithin 3 weeks of making (Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria that Grow Aerobically, 3rd Edition,NCCLS, Wayne, Pa., 1997).

Previous preliminary studies have shown that this gemifloxaxin is veryactive against Haemophilus and Moraxella (Cormican, et al., Antimicrob.Agents Chemother. 41:204-211, 1997; Hohl, et al., Clin. Microbiol.Infect. 4:280-284, 1998; and Oh, et al., Antimicrobial Agents Chemother.40:1564-1568, 1996).

A further embodiment provided herein is based in part on a significantdiscovery made using a gemifloxacin compound against nine rare clinicalstrains of Haemophilus influenzae from Europe with increasedciprofloxacin MICs were tested for in vitro activity (MICs) ofgemifloxacin (SB-265805), ciprofloxacin, levofloxacin, sparfloxacin,grepafloxacin and trovafloxacin and checked for mutations in gyrA, parC,gyrB and parE, demonstrating the activity of the gemifloxacin compoundused was superior to a number of quinolones as described in more detailherein. Gemifloxacin compounds are valuable compounds for the treatmentof infections caused by a range of Haemophilus influenzae strains,including those resistant to usual oral therapy, thereby filling anunmet medical need.

SUMMARY OF THE INVENTION

I. Pneumococcal Pathogens

An object of the invention is a method for modulating metabolism ofpneumococcal pathogenic bacteria comprising the step of contactingpneumococcal pathogenic bacteria with an antibacterially effectiveamount of a composition comprising a quinolone, particularly agemifloxacin compound, or an antibacterially effective derivativethereof.

A further object of the invention is a method wherein said pneumococcalpathogenic bacteria is selected from the group consisting of: bacteriacomprising a mutation in a quinolone resistance-determining region(QRDR) of parC, gyrA, parE, and/or gyrb; bacteria comprising a mutationin ParC at S79-F or Y, D83-N, R95-C, or K137-N; bacteria comprising amutation in gyrA at S83-A, C, F, or Y; E87-K; or S116-G; bacteriacomprising a mutation in parE at D435-N or I460-V; bacteria comprising amutation in gyrB at D435-N or E474-K; bacteria comprising at least fourmutations in a QRDR or parC, gyrA, parE, and gyrB; bacteria comprising amutation in a quinolone resistance-determining region (QRDR) of parC,gyrA, parE, and/or gyrB; bacteria that are ciprofloxacin-resistant,levofloxacin-resistant, sparfloxacin-resistant, grepafloxacin-resistant,or trovafloxacin-resistant, or a combination thereof, that comprise amutation in ParC at S79-F or Y, D83-N, R95-C, or K137-N; bacteria thatare ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in gyrA at S83-A, C, F, or Y; E87-K; or S116-G; bacteria thatare ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in parE at D435-N or I460-V; bacteria that areciprofloxacin-resistant, levofloxacin-resistant, sparfloxacin-resistant,grepafloxacin-resistant, or trovafloxacin-resistant, or a combinationthereof, that comprise a mutation in gyrB at D435-N or E474-K; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise atleast four mutations in a QRDR or parC, gyrA, parE, and gyrB; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in a quinolone resistance-determining region (QRDR) of parC,gyrA, parE, and/or gyrB; Streptococcus pneumoniae bacteria comprising amutation in ParC at S79-F or Y, D83-N, R95-C, or K137-N; Streptococcuspneumoniae bacteria comprising a mutation in gyrA at S83-A, C, F, or Y;E87-K; or S116G; Streptococcus pneumoniae bacteria comprising a mutationin pare at D435-N or I460-V; Streptococcus pneumoniae bacteriacomprising a mutation in gyrB at D435-N or E474-K; Streptococcuspneumoniae bacteria comprising at least four mutations in a QRDR orparC, gyrA, parE, and gyrB; and Streptococcus pneumoniae bacteriacomprising a mutation in a quinolone resistance-determining region(QRDR) of parC, gyrA, parE, and/or gyrB.

Also provided by the invention is a method of treating or preventing abacterial infection by pneumococcal pathogenic bacteria comprising thestep of administering an antibacterially effective amount of acomposition comprising a quinolone, particularly a gemifloxacin compoundto a mammal suspected of having or being at risk of having an infectionwith pneumococcal pathogenic bacteria.

A preferred method is provided wherein said modulating metabolism isinhibiting growth of said bacteria or killing said bacteria.

A further preferred method is provided wherein said contacting saidbacteria comprises the further step of introducing said composition intoa mammal, particularly a human.

Further preferred methods are provided by the invention wherein saidbacteria is selected from the group consisting of: bacteria comprising amutation in a quinolone resistance-determining region (QRDR) of parC,gyrA, parE, and/or gyrB; bacteria comprising a mutation in ParC at S79-For Y, D83-N, R95-C, or K137-N; bacteria comprising a mutation in gyrA atS83-A, C, F, or Y; E87-K; or S116-G; bacteria comprising a mutation inparE at D435-N or I460-V; bacteria comprising a mutation in gyrB atD435-N or E474-K; bacteria comprising at least four mutations in a QRDRor parC, gyrA, parE, and gyrB; bacteria comprising a mutation in aquinolone resistance-determining region (QRDR) of parC, gyrA, parE,and/or gyrB; bacteria that are ciprofloxacin-resistant,levofloxacin-resistant, sparfloxacin-resistant, grepafloxacin-resistant,or trovafloxacin-resistant, or a combination thereof, that comprise amutation in ParC at S79-F or Y, D83-N, R95-C, or K137-N; bacteria thatare ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in gyrA at S83-A, C, F, or Y; E87-K; or S116-G; bacteria thatare ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in pare at D435-N or I460-V; bacteria that areciprofloxacin-resistant, levofloxacin-resistant, sparfloxacin-resistant,grepafloxacin-resistant, or trovafloxacin-resistant, or a combinationthereof, that comprise a mutation in gyrB at D435-N or E474-K; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise atleast four mutations in a QRDR or parC, gyrA, parE, and gyrB; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in a quinolone resistance determining region (QRDR) of parC,gyrA, parE, and/or gyrB; Streptococcus pneumoniae bacteria comprising amutation in ParC at S79-F or Y, D83-N, R95-C, or K137-N; Streptococcuspneumoniae bacteria comprising a mutation in gyrA at S83-A, C, F, or Y;E87-K; or S116-G; Streptococcus pneumoniae bacteria comprising amutation in parE at D435-N or I460-V; Streptococcus pneumoniae bacteriacomprising a mutation in gyrB at D435-N or E474-K; Streptococcuspneumoniae bacteria comprising at least four mutations in a QRDR orparC, gyrA, parE, and gyrB; and Streptococcus pneumoniae bacteriacomprising a mutation in a quinolone resistance-determining region(QRDR) of parc, gyrA, parE, and/or gyrB.

Also provided is a method for modulating the activity of a topoisomerasecomprising a mutation in a quinolone resistance-determining region(QRDR) of parC, gyrA or parE or gyrB.

It is preferred in the methods of the invention that said mutation inParC is at S79-F or Y, D83-N, R95-C, or K137-N; said mutation in gyrA isat S83-A, C, F, or Y; E87-K; or S116-G; said mutation in parE is atD435-N or I460-V; or said mutation in gyrB is at D435-N or E474-K.

An object of the invention is a method for modulating metabolism ofquinolone-resistant pneumococcal pathogenic bacteria comprising the stepof contacting quinolone-resistant pneumococcal pathogenic bacteria withan antibacterially effective amount of a composition comprising aquinolone, particularly a gemifloxacin compound, or an antibacteriallyeffective derivative thereof.

A further object of the invention is a method wherein saidquinolone-resistant pneumococcal pathogenic bacteria is selected fromthe group consisting of: a pneumococcal strain comprising a mutation inthe quinolone resistance-determining region (QRDR) of parC and/or gyrA;a pneumococcal strain comprising a mutation in parC said mutationcomprising S79-F and/or Y, D83-G and/or N, N91-D, R95-C, and/or K137-N;a pneumococcal strain comprising a mutation in gyrA said mutationcomprising S81-A, C, F, or Y; E85-K; and/or S114-G; a pneumococcalstrain comprising a mutation in parE said mutation comprising D43 5-Nand/or I460-V; a pneumococcal strain comprising a mutation in gyrB saidmutation comprising D435-N and/or E474-K; a pneumococcal straincomprising a mutation in comprising three or four mutations in a QRDRsof parC, gyrA,parE, and/or gyrB; a pneumococcal strain comprising amutation in comprising three or four mutations in a QRDRs of parC,gyrA,parE, and/or gyrB, any of which are resistant to ciprofloxacin,levofloxacin, or sparfloxacin; and a pneumococcal strain comprising amutation in comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB, any of which also comprising an efflux mechanism ofquinolone resistance.

Also provided by the invention is a method of treating or preventing abacterial infection by quinolone-resistant pneumococcal pathogenicbacteria comprising the step of administering an antibacteriallyeffective amount of a composition comprising a quinolone, particularly agemifloxacin compound to a mammal suspected of having or being at riskof having an infection with quinolone-resistant pneumococcal pathogenicbacteria.

Further preferred methods are provided by the invention wherein saidbacteria is selected from the group consisting of: a pneumococcal straincomprising a mutation in the quinolone resistance-determining region(QRDR) of parC and/or gyrA; a pneumococcal strain comprising a mutationin parC said mutation comprising S79-F and/or Y, D83-G and/or N, N91-D,R95-C, and/or K137-N; a pneumococcal strain comprising a mutation ingyrA said mutation comprising S81-A, C, F, and/or Y; E85-K; and/orS114-G; a pneumococcal strain comprising a mutation in parE saidmutation comprising D435-N and/or I460-V; a pneumococcal straincomprising a mutation in gyrB said mutation comprising D435-N and/orE474-K; a pneumococcal strain comprising a mutation in comprising threeor four mutations in a QRDRs of parC, gyrA, parE, and/or gyrB; apneumococcal strain comprising a mutation in comprising three or fourmutations in a QRDRs of parC, gyrA, parE, and/or gyrB, any of which areresistant to ciprofloxacin, levofloxacin, or sparfloxacin; and apneumococcal strain comprising a mutation in comprising three or fourmutations in a QRDRs of parC, gyrA, parE, and/or gyrB, any of which alsocomprising an efflux mechanism of quinolone resistance.

II. Haemophilus Pathogens

An object of the invention is a method for modulating metabolism of arare Haemophilus influenzae strain comprising the step of contacting arare Haemophilus influenzae strain with an antibacterially effectiveamount of a composition comprising a quinolone, particularly agemifloxacin compound, or an antibacterially effective derivativethereof.

A further object of the invention is a method wherein said rarepathogenic H. influenzae strain is selected from the group consistingof: bacteria comprising a mutation set forth in Table 11 or 12; aHaemophilus influenzae strain set forth in Table 11 or 12; bacteria ofthe genus Haemophilus comprising a mutation set forth in Table 11 or 12;and bacteria of the species Haemophilus influenzae comprising a mutationset forth in Table 11 or 12.

Also provided by the invention is a method of treating or preventing abacterial infection by a rare pathogenic H. influenzae strain comprisingthe step of administering an antibacterially effective amount of acomposition comprising a quinolone, particularly a gemifloxacin compoundto a mammal suspected of having or being at risk of having an infectionwith a rare pathogenic H. influenzae strain.

A preferred method is provided wherein said modulating metabolism isinhibiting growth of said bacteria or killing said bacteria.

A further preferred method is provided wherein said contacting saidbacteria comprises the further step of introducing said composition intoa mammal, particularly a human.

Further preferred methods are provided by the invention wherein saidbacteria is selected from the group consisting of: bacteria comprising amutation set forth in Table 11 or 12; a Haemophilus influenzae strainset forth in Table 11 or 12; bacteria of the genus Haemophiluscomprising a mutation set forth in Table 11 or 12; and bacteria of thespecies Haemophilus influenzae comprising a mutation set forth in Table11 or 12.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

I. Pneumococcal Pathogens

The present invention provides, among other things, methods for using acomposition comprising a quinolone, particularly a gemifloxacin compoundagainst a number of pathogenic bacteria including, for example, strainsof Streptococcus pneumoniae and Haemophilus influenzae.

The present invention firther provides methods for using a compositioncomprising a quinolone, particularly a gemifloxacin compound against aquinolone-resistant pneumococcal strain, particularly a straincomprising a mutation in the quinolone resistance-determining region(QRDR) of parC and/or gyrA; a pneumococcal strain comprising a mutationin parC said mutation comprising S79-F and/or Y, D83-G and/or N, N91-D,R95-C, and/or K137-N; a pneumococcal strain comprising a mutation ingyrA said mutation comprising S81-A, C, F, and/or Y; E85-K; and/orS114-G; a pneumococcal strain comprising a mutation in parE saidmutation comprising D435-N and/or I460-V; a pneumococcal straincomprising a mutation in grB said mutation comprising D435-N and/orE474-K; a pneumococcal strain comprising a mutation in comprising threeor four mutations in a QRDRs of parC, gyrA, parE, and/or gyrB; apneumococcal strain comprising a mutation in comprising three or fourmutations in a QRDRs of parC, gyrA,parE, and/or gyrB, any of which areresistant to ciprofloxacin, levofloxacin, or sparfloxacin; and apneumococcal strain comprising a mutation in comprising three or fourmutations in a QRDRs of parC, gerA, parE, and/or gyrB, any of which alsocomprising an efflux mechanism of quinolone resistance.

As used herein “gemifloxacin compound(s)” means a compound havingantibacterial activity described in patent application PCT/KR98/00051published as WO 98/42705, or patent application EP 688772.

Previous studies have shown gemifloxacin to be 32 to 64 fold more activethan ciprofloxacin, ofloxacin, sparfloxacin and trovafloxacin againstmethicillin-susceptible and -resistant Staphylococcus aureus,methicillin-resistant Staphylococcus epideridis and S. pneumoniae.Gemifloxacin was also highly active against most members of the familyEnterobacteriaceae, with activity was more potent than those ofsparfloxacin and ofloxacin and comparable to that of ciprofloxacin.Gemifloxacin was the most active agent against Gram-positive speciesresistant to other quinolones and glycopeptides. Gemifloxacin haslimited activity against anaerobes (Cormican, et al., Antimicrob. AgentsChemother. 41:204-211, 1997; Hohl, et al., Clin. Microbiol. Infect.4:280-284, 1998; Oh, et al., Antimicrob. Agents Chemother. 40:1564-1568,1996).

This invention was based, in part, on analyses evaluating thecomparative activity of gemifloxacin against various pneumococcalpathogens. In these analyses, gemifloxacin gave the lowest quinoloneMICs against all pneumococcal strains tested followed by trovafloxacin,grepafloxacin, sparfloxacin, levofloxacin and ciprofloxacin. MICs weresimilar to those described previously (Cormican, et al., Antimicrob.Agents Chemother. 41:204-211, 1997; Hohl, et al., Clin. Microbiol.Infect. 4:280-284, 1998; Oh, et al., Antimicrob. Agents Chemother.40:1564-1568, 1996). Additionally, gemifloxacin gave significantly lowerMICs against highly quinolone resistant pneumococci, irrespective ofquinolone resistance mechanism. This was the case in double mutants withmutations in both parC and gyrA, strains which have previously beenshown to be highly resistant to other quinolones, as well as for strainswith an efflux mechanism (Brenwald, et al., Antimicrob. AgentsChemother. 42:2032-2035, 1998; and Pan et al., Antimicrob. AgentsChemother. 40:2321-2326, 1996). MICs of non-quinolone agents weresimilar to those described previously (M. R. Jacobs, Clin. Infect. Dis.15:119-127, 1992; Jacobs, et al., Rev. Med. Microbiol. 6:77-93, 1995;Pankuch, et al., J. Antimicrob. Chemother. 35:883-888, 1995).

Gemifloxacin also showed good killing against the 12 strains tested,including the two quinolone resistant strains. At ≦0.5 μg/ml,gemifloxacin was bactericidal against all 12 strains. Killing ratesrelative to MICs were similar to those of other quinolones, withsignificant killing occurring earlier than β-lactams and macrolides.Kill kinetics of quinolone and non-quinolone compounds in the analysesdescribed herein were similar to those described previously (Pankuch, etal., Antimicrob. Agents Chemother. 38:2065-2072, 1994; Pankuch et al.,Antimicrob. Agents Chemother. 40:1653-1656, 1996; and Visalli, et al.,Antimicrob. Agents Chemother. 40:362-366, 1996). Gemifloxacin also gave,together with the other quinolones tested, significant PAEs against all6 strains tested, including the one quinolone resistant strain. Thehigher ciprofloxacin PAE at both exposure concentrations is of nosignificance, because, with an MIC of 32 μg/ml, 5× and 10×MIC are notclinically achievable with this strain. PAE values for quinolones andmacrolides were similar to those described previously (Fuursted, et al.,Antimicrob. Agents Chemother. 41:781-784, 1997; Licata, et al.,Antimicrob. Agents Chemother. 41:950-955, 1997; Spangler, et al.,Antimicrob. Agents Chemother. 41:2173-2176, 1997; and Spangler, et al.,Antimicrob. Agents Chemother. 42:1253-1255, 1998). It is generallyaccepted that quinolones have similar PAEs against pneumococci.

In summary, gemifloxacin was the most potent quinolone tested by MIC andtime-kill against both quinolone susceptible and resistant pneumococciand, similar to other quinolones, gave PAEs against quinolonesusceptible strains. The incidence of quinolone resistant pneumococci iscurrently very low. However, this situation may change with theintroduction of broad-spectrum quinolones into clinical practice, and inparticular in the pediatric population, leading to selection ofquinolone resistant strains (Davies, et al., Antimicrob. AgentsChemother. 43:1177-1182, 1999). Gemifloxacin is a promising newantipneumococcal agent against pneumococci, irrespective of theirsusceptibility to quinolones and other agents. Clinical studies will benecessary in order to validate this hypothesis.

Results of agar dilution MIC testing of the 207 strains withciprofloxacin MICs ≦4.0 μg/ml are presented in Table 1. MIC_(50/90)values (μg/ml) were as follows: gemifloxacin, 0.03/0.06; ciprofloxacin,1.0/4.0; levofloxacin, 1.0/2.0; sparfloxacin, 0.5/0.5; grepafloxacin,0.125/0.5; trovafloxacin, 0.125/0.25; amoxicillin, 0.016/0.06(penicillin susceptible), 0.125/1.0 (penicillin intermediate), 2.0/4.0(penicillin resistant); cefuroxime, 0.03/0.25 (penicillin susceptible),0.5/2.0 (penicillin intermediate), 8.0/16.0 (penicillin resistant);azithromycin, 0.125/0.5 (penicillin susceptible), 0.125/>128.0(penicillin intermediate), 4.0/>128.0 (penicillin resistant);clarithromycin, 0.03/0.06 (penicillin susceptible), 0.03/32.0(penicillin intermediate), 2.0/>128.0 (penicillin resistant). Against 28strains with ciprofloxacin MICs ≧8 μg/ml, gemifloxacin had the lowestMICs (0.03-1.0 μg/ml, MIC₉₀ 0.5 μg/ml), compared with MICs rangingbetween 0.25 to >32.0 μg/ml)(MIC₉₀s 4.0→32.0 μg/ml) for the otherquinolones, with trovafloxacin, grepafloxacin, sparfloxacin andlevofloxacin, in ascending order, giving the next lowest MICs (Table 2).Mechanisms of quinolone resistance are presented in Tables 3 and 4. Ascan be seen, quinolone resistance was associated with mutations in thequinolone resistance-determining region (QRDR) of parC, gyrA, parE,and/or gyrB. Mutations in ParC were at S79-F or Y, D83-N, R95-C, orK137-N. Mutations in gyrA were at S83-A, C, F, or Y; E87-K; or S116-G.Twenty one strains had a mutation in parE at D435-N or I460-V. Only twostrains had a mutation in gyrB at D435-N or E474-K. Twenty strains had atotal of three or four mutations in the QRDRs or parC, gyrA, parE, andgyrB (Table 3). Amongst these 20 strains all were resistant tociprofloxacin (MICs >8 μg/ml), levofloxacin (MICs >4 μg/ml), andsparfloxacin (MICs >1 μg/ml); 19 were resistant to grepafloxacin(MICs >1 μg/ml); and 10 were resistant to trovafloxacin (MICs >2 μg/ml),yet gemifloxacin MICs were <0.5 μg/ml in 18 of the strains (Table 2).

In the presence of reserpine 23 strains had lower ciprofloxacin MICs(2-16×); 13 strains had lower gemifloxacin MICs (2-4×); 7 strains hadlower levofloxacin MICs (2-4×); 3 strains bad lower grepafloxacin MICs(2×); and one strain had lower sparfloxacin MICs (2×), suggesting thatan efflux mechanism contributed to the raised MICs in some cases (Table4).

Microbroth dilution MIC results of the 12 strains tested by time-killare presented in Table 5. Microdilution MICs were all within onedilution of agar MICs. For the two quinolone resistant strains (bothpenicillin susceptible), gemifloxacin microbroth MICs were 0.5 and 0.25μg/ml, respectively. Time-kill results (Table 6) showed thatlevofloxacin at the MIC, gemifloxacin and sparfloxacin at 2×MIC andciprofloxacin, grepafloxacin and trovafloxacin at 4×MIC, werebactericidal after 24 h. Various degrees of 90% and 99% killing by allquinolones was detected after 3 h. Gemifloxacin and trovafloxacin wereboth bactericidal at the microbroth MIC for the two quinolone resistantpneumococcal strains. Gemifloxacin was uniformly bactericidal after 24 hat ≦0.5 μg/ml. Amoxicillin, at 2×MIC and cefuroxime at 4×MIC, werebactericidal after 24 h, with some degree of killing at earlier timeperiods. By contrast, macrolides gave slower killing against the 7susceptible strains tested, with 99.9% killing of all strains at 2-4×MICafter 24 hours.

For the five quinolone susceptible strains tested for PAE, MICs weresimilar to those obtained by microdilution, with gemifloxacin havingMICs of 0.25 μg/ml against the quinolone resistant strain (MICs of otherquinolones 4-32 μg/ml). PAEs (h)(10×MIC) for the 5 quinolone susceptiblestrains ranged between 0.4-1.6 for gemifloxacin; 0.5-1.5 h forciprofloxacin (except for the quinolone resistant strain which gave aciprofloxacin PAE of 6.3); 0.9-2.3 (levofloxacin); 0.3-2.0(sparfloxacin); 0.3-2.6 (grepafloxacin); 1.3-3.0 (trovafloxacin). At5×MIC, PAEs (h) for the quinolone resistant strain were 0.9(gemifloxacin); 3.7 (ciprofloxacin); 1.3 (levofloxacin); 1.5(sparfloxacin); 1.5 (grepafloxacin); 1.3 (trovafloxacin). PAEs fornon-quinolone compounds (10×MIC) ranged between 0.3-5.8 (amoxicillin);0.8-2.9 (cefuroxime); 1.3-3.0 (azithromycin); 1.8-4.5 (clarithromycin).

TABLE 1 Agar dilution MICs (μg/ml) of 207 quinolone susceptiblestrains^(a) Drug MIC range MIC₅₀ MIC₉₀ Penicillin Penicillin S≦0.008-0.06 0.016 0.03 Penicillin I 0.125-1.0 0.25 1.0 Penicillin R2.0-16.0 4.0 4.0 Gemifloxacin Penicillin S ≦0.008-0.125 0.03 0.03Penicillin I ≦0.008-0.25 0.03 0.06 Penicillin R 0.004-0.125 0.03 0.06Ciprofloxacin Penicillin S 0.25-4.0 1.0 2.0 Penicillin I 0.25-4.0 1.02.0 Penicillin R 0.5-4.0 1.0 4.0 Levofloxacin Penicillin S 0.125-4.0 1.02.0 Penicillin I 0.5-4.0 1.0 2.0 Penicillin R 1.0-2.0 1.0 2.0Sparfloxacin Penicillin S ≦0.03-1.0 0.5 1.0 Penicillin I 0.06-2.0 0.50.5 Penicillin R 0.06-1.0 0.5 0.5 Grepafloxacin Penicillin S ≦0.03-1.00.125 0.5 Penicillin I ≦0.03-0.5 0.125 0.5 Penicillin R ≦0.03-0.5 0.250.5 Trovafloxacin Penicillin S 0.03-0.5 0.125 0.25 Penicillin I0.016-1.0 0.125 0.25 Penicillin R 0.03-0.25 0.125 0.25 AmoxicillinPenicillin S ≦0.008-0.25 0.016 0.06 Penicillin I 0.016-4.0 0.125 1.0Penicillin R 0.5-8.0 2.0 4.0 Cefuroxime Penicillin S ≦0.008-2.0 0.030.25 Penicillin I 0.125-8.0 0.5 2.0 Penicillin R 0.5-32.0 8.0 16.0Azithromycin Penicillin S ≦0.008->128.0 0.125 0.5 Penicillin I≦0.008->128.0 0.125 >128.0 Penicillin R 0.03->128.0 4.0 >128.0Clarithromycin Penicillin S ≦0.008->128.0 0.03 0.06 Penicillin I≦0.008->128.0 0.03 32.0 Penicillin R 0.008->128.0 2.0 >128.0^(a)Ciprofloxacin MICs ≦4.0 μg/ml.

TABLE 2 Quinolone agar dilution MICs (μg/ml) of 28 ciprofloxacinresistant strains^(a) Quinolone MIC range MIC₅₀ MIC₉₀ Gemifloxacin0.3-1.0   0.25 0.5 Ciprofloxacin 8.0->32.0 16.0 >32.0 Levofloxacin4.0->32.0 16.0 >32.0 Sparfloxacin 0.25->32.0  8.0 16.0 Grepafloxacin0.5-16.0  4.0 8.0 Trovafloxacin 0.25-8.0    1.0 4.0 ^(a)CiprofloxacinMICs ≧8.0 μg/ml.

TABLE 3 Correlation of quinolone MIC (μg/ml) and mutation in quinoloneresistant strains. Gemifloxaci Ciprofloxaci Levofloxaci SparfloxacinGrepafloxaci Trovafloxac Mutation Strain n^(a) n^(a) n^(a) ^(a) n^(a)in^(a) ParC ParE GyrA GryB 1 0.03 16 8 4 4 2 S79-F I460-V S83-F None 20.06 8 4 0.5 0.5 0.25 S79-Y I460-V None None 3 0.06 8 4 1 0.5 0.25 D83-NI460-V S83-F None 4 0.06 8 4 1 1 0.25 S79-F I460-V S83-F None 5 0.125 88 1 1 0.25 R95-C D435-N S83-F None 6 0.125 8 8 8 2 2 S79-Y I460-V E87-KNone 7 0.125 8 8 1 1 0.5 None I460-V None None 8 0.125 8 8 1 1 0.5 S79-YNone None None 9 0.125 8 8 2 1 1 S79-Y None None None 10 0.125 8 8 4 2 1S79-F I460-V S83-C None 11 0.125 8 8 4 4 1 S79-F I460-V S83-F None 120.125 >32 16 1 4 1 S79-F I460-V None D435-N 13 0.25 16 8 8 2 1 S79-FI460-V None EA74-K 14 0.25 16 16 8 4 1 S79-F I460-V S83-F None 15 0.2516 16 8 4 1 79-F I460-V S83-F None 16 0.25 16 16 8 4 2 S79-F I460-VS83-F None 17 0.25 16 16 8 4 2 D83-N None S83-F None 18 0.25 16 16 8 4 2S79-F I460-V S83-F None 19 0.25 16 16 8 4 2 S79-F I460-V S83-F None 200.25 32 16 8 4 2 S79-F I460-V E87-K None 21 0.25 32 16 8 4 1 S79-FI460-V S83-F None 22 0.25 32 16 8 4 2 S79-F I460-V S83-Y None 23 0.25 3216 8 8 2 S79-Y None S83-A None 24 0.5 32 32 16 8 4 S79-F I460-V S83-FNone 25 0.5 32 32 16 8 4 None S83-F None 26 0.5 >32 >32 >32 8 4 S79-FNone S83-Y None 27 1 >32 >32 >32 16 8 S79-Y I460-V S83-F None 281 >32 >32 >32 16 8 None S83-F None S116-G ^(a)MIC(μg/ml).

TABLE 4 Efflux mechanisms in quinolone resistant pneumococci StrainGemifloxacin Ciprofloxacin Levofloxacin Sparfloxacin GrepafloxacinTrovafloxacin 1 2 X^(a) 2 X — — — — 2 — 8 x — — — — 3 — — — — — — 4 — —— — — — 5 2 X 4 X — 2 X 2 X — 6 — 2 X — — — — 7 2 X 4 X 2 X — — — 8 2 X2 X — — — — 9 4 X 8 X — — — — 10 — 2 X — — — — 11 2 X 16 X  4 X — — — 122 X 4 X 2 X — 2 X — 13 — 2 X — — — — 14 — — — — — — 15 — — — — — — 16 2X 4 X — — — — 17 2 X 4 X — — — — 18 — 2 X — — — — 19 — 2 X — — — — 20 —2 X — — — — 21 — 2 X — — — — 22 — 2 X — — — — 23 2 X 8 X 2 X — — — 24 —2 x — — — — 25 2 x 4 x 2 x — — — 26 2 x 4 x 2 x — — — 27 — — — — — — 284 x 8 x 4 x — 2 X —

TABLE 5 Microdilution MICs of 12 strains tested by time-kill Drug 1(S)^(a) 2 (S) 3 (S)^(b) 4 (S)^(b) 5 (I) 6 (I) 7 (I) 8 (I) 9 (R) 10 (I)11 (R) 12 (R) Penicillin G 0.06 0.03 0.016 0.016 0.25 0.25 1 0.5 4 2 4 4Gemifloxacin 0.016 0.016 0.5 0.25 0.03 0.016 0.016 0.016 0.03 0.0160.016 0.03 Ciprofloxacin 1 0.5 32 32 2 1 4 0.5 1 1 2 1 Levofloxacin 2 132 32 1 2 1 1 2 2 1 2 Sparfloxacin 0.125 0.25 32 16 0.5 0.25 0.25 0.250.5 0.25 0.25 0.5 Grepafloxacin 0.06 0.06 16 8 0.125 0.125 0.125 0.1250.25 0.125 0.125 0.25 Trovafloxacin 0.06 0.06 8 4 0.06 0.06 0.06 0.1250.125 0.06 0.06 0.125 Amoxicillin 0.016 0.016 0.008 0.008 0.03 0.1250.125 0.06 1 1 2 2 Cefuroxime 0.5 0.25 0.016 0.016 0.5 0.5 0.5 0.25 20.5 4 2 Azithromycin 0.008 0.06 >64 0.125 >64 0.03 0.125 0.125 >64 >640.125 >64 Clarithromycin 0.008 0.03 >64 0.03 32 0.008 0.016 0.03 >64 >640.03 >64 ^(a)S = penicillin susceptible; I = penicillin intermediate; R= penicillin resistant. ^(b)Quinolone-resistant.

TABLE 6 Time-kill results of 12 pneumococcal strains 3h 6h 12h 24h Drug−1^(a) −2^(a) −3^(a) −1 −2 −3 −1 −2 −3 −1 −2 −3 Gemifloxacin 8 × MIC10^(b) 2 0 12 8 2 12 12 9 12 12 12 4 × MIC 9 1 0 12 8 0 12 12 8 12 12 122 × MIC 6 0 0 12 7 0 12 11 8 12 12 12 MIC 4 1 0 11 2 0 12 8 3 12 10 80.5 × MIC 1 0 0 4 0 0 3 0 0 2 2 0 0.25 × MIC 0 0 0 0 0 0 0 0 0 0 0 0Ciprofloxacin 8 × MIC 10 8 2 12 11 6 12 12 10 12 12 12 4 × MIC 9 6 1 1210 5 12 12 10 12 12 12 2 × MIC 9 4 0 12 8 2 12 12 6 12 12 11 MIC 4 0 0 83 0 10 9 3 11 10 6 0.5 × MIC 0 0 0 1 1 0 2 1 0 2 1 0 0.25 × MIC 0 0 0 00 0 0 0 0 0 0 0 Levofloxacin 8 × MIC 11 3 0 12 9 4 12 12 10 12 12 12 4 ×MIC 10 4 0 12 9 1 12 12 8 12 12 12 2 × MIC 10 2 0 12 9 1 12 12 9 12 1212 MIC 9 1 0 12 6 0 12 11 7 12 12 12 0.5 × MIC 4 1 0 8 1 0 7 3 0 8 7 50.25 × MIC 0 0 0 0 0 0 0 0 0 0 0 0 Sparfloxacin 8 × MIC 10 2 0 12 9 4 1212 9 12 12 12 4 × MIC 9 1 0 12 8 0 12 11 8 12 12 12 2 × MIC 8 1 0 12 4 012 10 5 12 12 12 MIC 4 0 0 8 2 0 11 9 4 11 11 10 0.5 × MIC 1 0 0 5 1 0 40 0 6 4 1 0.25 × MIC 0 0 0 0 0 0 0 0 0 1 1 1 Grepafloxacin 8 × MIC 8 2 112 5 2 12 11 7 12 12 12 4 × MIC 6 0 0 12 4 0 12 10 5 12 12 12 2 × MIC 30 0 9 1 0 10 8 1 11 10 9 MIC 1 0 0 4 1 0 7 3 0 8 5 3 0.5 × MIC 0 0 0 0 00 1 0 0 1 1 0 0.25 × MIC 0 0 0 0 0 0 0 0 0 0 0 0 Trovafloxacin 8 × MIC12 3 0 12 10 1 12 12 9 12 12 12 4 × MIC 9 2 0 12 9 1 12 10 8 12 12 12 2× MIC 5 1 0 11 4 0 12 11 7 11 11 11 MIC 4 0 0 6 2 0 7 4 1 6 1 1 0.5 ×MIC 0 0 0 0 0 0 0 0 0 0 0 0 0.25 × MIC 0 0 0 0 0 0 0 0 0 0 0 0Amoxicillin 8 × MIC 9 4 0 11 8 3 12 12 10 12 12 12 4 × MIC 7 2 0 12 7 012 12 9 12 12 12 2 × MIC 6 2 0 11 5 1 12 11 8 12 12 12 MIC 4 0 0 6 1 0 76 2 10 9 7 0.5 × MIC 0 0 0 0 0 0 1 1 0 3 2 1 0.25 × MIC 0 0 0 0 0 0 0 00 1 0 0 Cefuroxime 8 × MIC 9 5 0 12 12 4 12 12 12 12 12 12 4 × MIC 9 3 012 11 1 12 12 11 12 12 12 2 × MIC 7 1 0 12 8 1 12 11 8 12 12 11 MIC 4 00 7 2 0 9 7 1 9 9 9 0.5 × MIC 3 0 0 1 0 0 0 0 0 1 1 0 0.25 × MIC 0 0 0 00 0 0 0 0 0 0 0 Azithromycin^(c) 8 × MIC 3 1 0 6 4 2 7 5 5 7 7 7 4 × MIC4 1 0 6 3 2 6 5 4 7 7 7 2 × MIC 3 1 0 4 2 2 5 5 4 7 7 5 MIC 2 0 0 3 2 25 5 2 7 6 5 0.5 × MIC 1 0 0 1 1 1 1 1 1 1 1 1 0.25 × MIC 0 0 0 0 0 0 0 00 0 0 0 Clarithro- mycin^(c) 8 × MIC 4 2 0 7 2 2 5 5 5 7 7 7 4 × MIC 3 20 7 2 2 5 5 5 7 7 7 2 × MIC 3 2 0 5 2 2 5 5 4 7 7 7 MIC 3 1 0 5 0 1 5 52 7 7 5 0.5 × MIC 1 0 0 3 0 0 1 1 1 4 3 1 0.25 × MIC 0 0 0 0 0 0 0 0 0 00 0 ^(a)ÄLog10 cfu/ml lower than 0 h. ^(b)No. strains tested. ^(c)Only 7strains with macrolide MICs ≦0.125 μg/ml were tested.

A further embodiment of the present invention is based, in part, onexperiments wherein in vitro activity of gemifloxacin was compared withthat of ciprofloxacin, levofloxacin, sparfloxacin, grepafloxacin andtrovafloxacin against 28 pneumococci with ciprofloxacin MICs ≧8 μg/ml.Gemifloxacin MICs (μg/ml) ranged between 0.03-1.0 (MIC_(50/90)0.25/0.5), compared with ciprofloxacin 8→32 (MIC_(50/90) 16/>32),levofloxacin 4→32 (MIC_(50/90) 16/>32), sparfloxacin 0.25→32(MIC_(50/90) 8/16), grepafloxacin 0.5-16 (MIC_(50/90) 4/8) andtrovafloxacin 0.25-8 (MIC_(50/90) 1.0/4.0). DNA sequence analysis showedthat all but one strain had a mutation in the quinoloneresistance-determining region (QRDR) of parC and/or gyrA. Mutations inparC were at S79-F or Y, D83-G or N, N91-D, R95-C, or K137-N. Mutationsin gyrA were at S81-A, C, F, or Y; E85-K; or S114-G. Twenty-one strainshad a mutation in parE at D435-N or I460-V. Only two strains had amutation in gyrB at D435-N or E474-K. Twenty-one strains had a total ofthree or four mutations in the QRDRs of parC, gyrA, pare, and gyrB. Ofthese 21 strains, all were resistant to ciprofloxacin (MIC ≧8 μg/ml),levofloxacin (MIC ≧4 μg/ml), and sparfloxacin (MIC ≧1 μg/ml); 20 wereresistant to grepafloxacin (MIC ≧1 μg/ml) and 11 were resistant totrovafloxacin (MIC ≧2 μ/ml), yet gemifloxacin MICs were ≦0.5 μg/ml in 19of the strains. In the presence of reserpine, 23 strains had lowerciprofloxacin MICs (2-16×), 13 strains had lower gemifloxacin MICs(2-4×), 7 strains had lower levofloxacin MICs (2-4×); 3 strains hadlower grepafloxacin MICs (2×) and one strain had lower sparfloxacin MICs(2×), indicating that an efflux mechanism contributed to the raised MICsin some cases. Results show that, irrespective of the mechanism ofquinolone resistance, gemifloxacin showed the greatest in vitro activityagainst all pneumococcal strains tested. Against 28 strains withciprofloxacin MICs ≧8 μg/ml, gemifloxacin had the lowest MICs (0.03-1.0μg/ml, MIC₉₀ 0.5 μg/ml), compared with MICs ranging between 0.25to >32.0 μg/ml (MIC₉₀ s4.0→32.0 μg/ml) for the other quinolones, withtrovafloxacin, grepafloxacin, sparfloxacin and levofloxacin, inascending order, giving the next lowest MICs (Table 7). Mechanisms ofquinolone resistance are presented in Tables 8 and 9. As can be seen,quinolone resistance was associated with mutations in the quinoloneresistance-determining region (QRDR) of parC, gyrA, parE and/or gyrB.Mutations in ParC were at S79-F or Y, D83-N, R95-C, or K137-N. Mutationsin gyrA were at S83-A, C, F, or Y; E87-K; or S116-G. Twenty-one strainshad a mutation in parE at D435-N or I460-V. Only two strains had amutation in grB at D435-N or E474-K. Twenty-one strains had a total ofthree or four mutations in the QRDRs of parC, gyrA, pare and gyrB (Table8). Amongst these 21 strains all were resistant to ciprofloxacin (MICs≧8 μg/ml), levofloxacin (MICs ≧4 μg/ml), and sparfloxacin (MICs ≧1μg/ml), 20 were resistant to grepafloxacin (MICs ≧1 μg/ml) and 11 wereresistant to trovafloxacin (MICs ≧2 μg/ml), yet gemifloxacin MICs were≦0.5 μg/ml in 19 of the strains (Table 8).

In the presence of reserpine 23 strains had lower ciprofloxacin MICs(2-16×), 13 strains had lower gemifloxacin MICs (2-4×), 7 strains badlower levofloxacin MICs (2-4×); 3 strains had lower grepafloxacin MICs(2×); and one strain bad lower sparfloxacin MICs (2×), indicating thatan efflux mechanism contributed to the raised MICs in some cases (Table9). Previous studies have shown gemifloxacin to be 32 to 64 fold moreactive than ciprofloxacin, ofloxacin, sparfloxacin and trovafloxacinagainst methicillin-susceptible and -resistant Staphylococcus aureus,methicillin-resistant Ataphylococcus epidennidis and S. pneumoniaeGemifloxacin was also highly active against most members of the familyEnterobacteriaceae, with activity which was more potent than those ofsparfloxacin and ofloxacin and comparable to that of ciprofloxacin.Gemifloxacin was the most active agent against Gram positive speciesresistant to other quinolones and glycopeptides. Gemifloxacin hasvariable activity against anaerobes, and is very active against the Grampositive group (Cormican, et al., Antimicrobiol. Agents Chemother. 41:204-211, 1997; Hohl, et al., Clin. Microbiol. Infect. 4:280-284, 1998;Oh, et al., Antimicrob. Agents Chenother. 40:1564-1568, 1996).

In our study, gemifloxacin gave significantly lower MICs against highlyquinolone-resistant pneumococci, irrespective of quinolone resistancemechanism. This was the case in double mutants with mutations in bothparC and gyrA, strains which have previously been shown to be highlyresistant to other quinolones, as well as for strains with an effluxmechanism (Pan, et al., Antimicrob. Agents Chemother. 40:2321-2326, 1996and Brenwald, et al., Antimicrob. Agents Chemother. 42:2032-2035, 1998).

In summary, gemifloxacin was the most potent quinolone tested againstquinolone resistant pneumococci. The incidence of quinolone-resistantpneumococci is currently very low. However, this situation may changewith the introduction of broad-spectrum quinolones into clinicalpractice, and in particular in the pediatric population, leading toselection of quinolone-resistant strains (Davies, et al., Antimicrob.Agents Chemother. 43:1177-1182, 1999). Results indicate that selectiveintroduction of quinolones such as gemifloxacin into the pediatricenvironment is predicated upon toxicologic studies. Additionally, if theincidence of quinolone-resistant pneumococci increases, gemifloxacinwill be a well-placed therapeutic option. Gemnifloxacin is a promisingnew antipneumococcal agent, irrespective of the strain's susceptibilityto quinolones and other agents.

TABLE 7 Quinolone Agar Dilution MICs (μg/ml) of 28Ciprofloxacin-Resistant Strains (MICs ≧8.0 μg/ml) Quinolone MIC rangeMIC₅₀ MIC₉₀ Gemifloxacin 0.3-1.0 0.25 0.5 Ciprofloxacin 8.0->32.016.0 >32.0 Levofloxacin 4.0->32.0 16.0 >32.0 Sparfloxacin 0.25->32.0 8.016.0 Grepafloxacin 0.5-16.0 4.0 8.0 Trovafloxacin 0.25-8.0 1.0 4.0

TABLE 8 Correlation of Quinolone MIC (μg/ml) and Mutation inQuinolone-Resistant Strains MIC (μg/ml) Gemifloxaci CiprofloxaciLevofloxaci Grepafloxaci Trovafloxac Mutation Strain n n n Sparfloxacinn in ParC ParE GyrA GyrB 1 0.03 16 8 4 4 2 S79-F I460-V S81-F None 20.06 8 4 0.5 0.5 0.25 S79-Y I460-V None None 3 0.06 8 4 1 0.5 0.25 D83-NI460-V S81-F None 4 0.06 8 4 1 1 0.25 S79-F I460-V S81-F None 5 0.125 88 1 1 0.25 R95-C D435-N S81-F None 6 0.125 8 8 8 2 2 S79-Y I460-V E85-KNone 7 0.125 8 8 1 1 0.5 None I460-V None None 8 0.125 8 8 1 1 0.5 S79-YNone None None 9 0.125 8 8 2 1 1 S79-Y None None None 10 0.125 8 8 4 2 1S79-F I460-V S81-C None 11 0.125 8 8 4 4 1 S79-F I460-V S81-F None 120.125 >32 16 1 4 1 S79-F I460-V None D435-N 13 0.25 16 8 8 2 1 S79-FI460-V None EA74-K 14 0.25 16 16 8 4 1 S79-F I460-V S81-F None 15 0.2516 16 8 4 1 S79-F I460-V S81-F None 16 0.25 16 16 8 4 2 S79-F I460-VS81-F None 17 0.25 16 16 8 4 2 D83-N None S81-F None 18 0.25 16 16 8 4 2S79-F I460-V S81-F None 19 0.25 16 16 8 4 2 S79-F I460-V S81-F None 200.25 32 16 8 4 2 S79-F I460-V E85-K None 21 0.25 32 16 8 4 1 S79-FI460-V S81-F None 22 0.25 32 16 8 4 2 S79-F I460-V S81-Y None 23 0.25 3216 8 8 2 S79-Y None S81-A None 24 0.5 32 32 16 8 4 S79-F I460-V S81-FNone 25 0.5 32 32 16 8 4 D83-G None S81-F None N91-D 26 0.5 >32 >32 >328 4 S79-F None S81-Y None 27 1 >32 >32 >32 16 8 S79-Y I460-V S81-F None28 1 >32 >32 >32 16 8 D83G None S81-F None N91-D S114-G

TABLE 9 Efflux Mechanisms in Quinolone-Resistant Pneumococci StrainGemifloxacin Ciprofloxacin Levofloxacin Sparloxacin GrepafloxacinTrovafloxacin 1 2 X^(a) 2 X — — — — 2 — 8 x — — — — 3 — — — — — — 4 — —— — — — 5 2 X 4 X — 2 X 2 X — 6 — 2 X — — — — 7 2 X 4 X 2 X — — — 8 2 X2 X — — — — 9 4 X 8 X — — — — 10 — 2 X — — — — 11 2 X 16 X 4 X — — — 122 X 4 X 2 X — 2 X — 13 — 2 X — — — — 14 — — — — — — 15 — — — — — — 16 2X 4 X — — — — 17 2 X 4 X — — — — 18 — 2 X — — — — 19 — 2 X — — — — 20 —2 X — — — — 21 — 2 X — — — — 22 — 2 X — — — — 23 2 X 8 X 2 X — — — 24 —2 x — — — — 25 2 x 4 x 2 x — — — 26 2 x 4 x 2 x — — — 27 — — — — — — 284 x 8 x 4 x — 2 X — ^(a)Number of dilutions decrease after incubationwith reserpine (see Materials and Methods). National Committee forClinical Laboratory Standards. 1997. Methods for dilution antimicrobialsusceptibility tests for bacteria that grow aerobically -- thirdedition; approved standard. NCCLS publication no. M7-A4. NationalCommittee for Clinical Laboratory Standards, Villanova, PA.

II. Haemophilus Pathogens

Nine quinolone-resistant H. influenzae strains yielded MIC₅₀s of 0.25μg/ml for gemifloxacin (highest MIC 1.0 μg/ml) compared to 1.0 μg/ml(highest MIC 4.0-8.0 μg/ml) for the other quinolones tested (Table 10).Mechanisms of quinolone resistance in the H. influenzae strains arepresented in Table 11. All nine strains had mutations at Ser 84 in GyrAwith Ser 84 to Leu, Phe, or Tyr observed. Additional mutations in GyrAat Asp 88 to Asn or Tyr, and Ala 117 to Glu were also observed in somestrains. Most strains also had at least one mutation in ParC (at Asp 83,Ser 84, Glu 88, Ser 133, or Asn 138) and ParE (at Gly 405, Asp 420, Ser458, or Ser 474). Strain 4 had an in-frame insertion in parE that led toan insertion of a Ser residue in between Ser 458 and Thr 459. Only onestrain had a mutation in GyrB (at Gln 468). The most resistant strain(strain 9) had double mutations in GyrA, ParC and ParE.

Previous studies have shown gemifloxacin to be 32-64 fold more activethan ciprofloxacin, ofloxacin, sparfloxacin and trovafloxacin againstmethicillin-susceptible and -resistant S. aureus, methicillin-resistantStaphylococcus epideridis and S. pneumoniae Gemifloxacin was also highlyactive against most members of the family Enterobacteriaceae, withactivity more potent than those of sparfloxacin and ofloxacin andcomparable to that of ciprofloxacin. Gemifloxacin was the most activeagent against Gram positive species resistant to other quinolones andglycopeptides. Gemifloxacin has variable activity against anaerobes andis very active against the Gram positive group (Cormican, et al.,Antimicrob. Agents Chemother. 41:204-211, 1997; Hohl, et al., Clin.Microbiol. Infect. 4:280-284, 1998; and Oh, et al., Antimicrob. AgentsChemother. 40:1564-1568, 1996. In the studies set forth herein, onlygemifloxacin gave MICs ≦1.0 μg/ml against the rare strains of H.influenzae with raised quinolone MICs. Previous studies (Bootsma, etal., J. Antimicrob. Chemother. 39:292-293, 1997; Georgiou, et al.,Antimicrob. Agents Chemother. 40:1741-1744, 1996; and Vila, et al.,Antimicrob. Agents Chemother. 43:161-162, 1999) have shown that theprimary target of quinolones in H. influenzae is GyrA; low-levelresistance is associated with a mutation in GyrA (Ser 84 or Asp 88) andhigh-level resistance with an additional mutation in ParC (Asp 83, Ser84 or Glu 88). Sequencing results from this study were in agreement withthe above previous reports, as all nine strains had at least onemutation in GyrA and the most resistant strains (ciprofloxacin MICs ≧1.0μg/ml) had an additional mutation in ParC. Mutations were found in GyrA(Ala 117) and ParC (Ser 133, Asn 138) that have not been previouslyreported. Provided herein is a novel examination of mutations in GyrBand ParE in H. influenzae: most strains had mutations in ParE, but onlyone strain in GyrB. Of particular interest was insertion of a serinebetween serine 458 and threonine 459 of ParE in one strain. It,therefore, appears that ParE is more important in quinolone resistancein H. influenzae than GyrB.

Results of this study indicate excellent activity of gernifloxacinagainst quinolone-resistant i H. influenzae (including those withmultiple mutations) by MIC. Because of the wide spectrum of activity ofgemifloxacin against other respiratory pathogens, such as pneumococci(including quinolone-resistant strains), Legionella, mycoplasmas andchlamydia, this compound represents an attractive alternative to otherquinolone and non-quinolone agents for empiric treatment ofcommunity-acquired respiratory tract infections.

TABLE 10 Quinolone MICs (μg/ml) for 9 Quinolone-Resistant Haemophilusinfluenzae strains Antimicrobial Range MIC₅₀ Gemifloxacin 0.03-1.0 0.25Ciprofloxacin 0.25-8.0 1.0 Levofloxacin 0.25-4.0 1.0 Sparfloxacin0.25-8.0 1.0 Grepafloxacin 0.25-4.0 1.0 Trovafloxacin 0.25-8.0 1.0

TABLE 11 Mechanisms of Resistance in Quinolone-Resistant Haemophilusinfluenzae strains MIC (μg/ml) Strain Gemi Cipro Levo Spar Grepa TrovaMutation 1 0.03 0.5 0.5 0.25 0.25 1 S133-A None S84-L None N138-S 20.125 0.25 0.25 0.25 0.5 0.25 NONE S458-L S84-F None 3 0.125 1 1 0.5 0.51 S84-I None S84-L None 4 0.25 1 0.5 0.25 2 0.5 D83-N S458-S-T459 S84-FQ468-R D88-N 5 0.25 1 1 1 1 1 E88-K G405-S S84-Y None 6 0.5 2 2 1 4S84-R D420-N S84-L None A117-E 7 0.5 2 2 1 1 4 S84-R D420-N S84-L NoneA117-E 8 0.5 2 2 1 1 4 S84-R D420-N S84-L None A117-E 9 1 8 4 8 4 8S84-R S458-A S84-F None N138-S S474-N D88-Y

TABLE 12 MICs (μg/ml) Mutation Strain Gem Cip Lev Spa Gre Tro GyrA ParCGyrB ParE 1 0.03 0.5 0.5 0.25 0.25 1 S84-L S133-A None None N138-S 20.125 0.25 0.25 0.25 0.5 0.25 S84-F None None S458-L 3 0.125 1 1 0.5 0.51 S84-L S84-I None None 4 0.25 1 0.5 0.25 2 0.5 S84-F D83-N None T459-SD88-N 5 0.25 1 1 1 1 1 S84-Y E88-K Q468-R G405-S 6 0.5 2 2 1 1 4 S84-LS84-R None D420-N A117-E 7 0.5 2 2 1 1 4 S84-L S84-R None D420-N A117-E8 0.5 2 2 1 1 4 S84-L S840-R None D420-N A117-E 9 1 8 4 8 4 8 S84-FS84-R None S458-A D88-Y N138-S S474-N

All strains had mutations at position 84 in gyrA, and the most R strainhad double mutations in gyrA, parC and parE. Strains with mutations atposition 84 in parC and gyrA plus mutations in parE were tro R. Gem hadthe lowest MICs against all strains irrespective of their mutationmechanism.

The invention provides a method for modulating metabolism of a rarepathogenic H. influenzae strain. Skilled artisans can readily choose arare pathogenic Haemophilus. influenzae strain or patients infected withor suspected to be infected with these organisms to practice the methodsof the invention. Alternatively, the bacteria useful in the methods ofthe invention may be those described herein.

The invention provides a method for modulating metabolism ofpneumococcal and Haemophilus pathogenic bacteria. Skilled artisans canreadily choose pneumococcal and Haemophilus pathogenic bacteria orpatients infected with or suspected to be infected with these organismsto practice the methods of the invention. Alternatively, the bacteriauseful in the methods of the invention may be those described herein.

The contacting step in any of the methods of the invention may beperformed in many ways that will be readily apparent to the skilledartisan. However, it is preferred that the contacting step is aprovision of a composition comprising a gemifloxacin compound to a humanpatient in need of such composition or directly to bacteria in culturemedium or buffer.

For example, when contacting a human patient or contacting said bacteriain a human patient or in vitro, the compositions comprising a quinolone,particularly a gemifloxacin compound, preferably pharmaceuticalcompositions may be administered in any effective, convenient mannerincluding, for instance, administration by topical, oral, anal, vaginal,intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal orintradermal routes among others.

It is also preferred that these compositions be employed in combinationwith a non-sterile or sterile carrier or carriers for use with cells,tissues or organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a compound ofthe invention, a quinolone, preferably a gemifloxacin compound, and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration.

Quinolone compounds, particularly gemifloxacin compounds andcompositions of the methods of the invention may be employed alone or inconjunction with other compounds, such as bacterial efflux pumpinhibitor compounds or antibiotic compounds, particularly non-quinolonecompounds, e.g., beta-actam antibiotic compounds.

In therapy or as a prophylactic, the active agent of a method of theinvention is preferably administered to an individual as an injectablecomposition, for example as a sterile aqueous dispersion, preferably anisotonic one.

Alternatively, the gemifloxacin compounds or compositions in the methodsof the invention may be formulated for topical application for examplein the form of ointments, creams, lotions, eye ointments, eye drops, eardrops, mouthwash, impregnated dressings and sutures and aerosols, andmay contain appropriate conventional additives, including, for example,preservatives, solvents to assist drug penetration, and emollients inointments and creams. Such topical formulations may also containcompatible conventional carriers, for example cream or ointment bases,and ethanol or oleyl alcohol for lotions. Such carriers may constitutefrom about 1% to about 98% by weight of the formulation; more usuallythey will constitute up to about 80% by weight of the formulation.

For administration to mammals, and particularly humans, it is expectedthat the antibacterially effective amount is a daily dosage level of theactive agent from 0.001 mg/kg to 10 mg/kg, typically around 0.1 mg/kg to1 mg/kg, preferably about 1 mg/kg. A physician, in any event, willdetermine an actual dosage that is most suitable for an individual andwill vary with the age, weight and response of the particularindividual. The above dosages are exemplary of the average case. Therecan, of course, be individual instances where higher or lower dosageranges are merited, and such are within the scope of this invention. Itis preferred that the dosage is selected to modulate metabolism of abacteria in such a way as to inhibit or stop growth of said bacteria orby killing said bacteria. The skilled artisan may identify this amountas provided herein as well as using other methods known in the art, e.g.by the application MIC tests.

A further embodiment of the invention provides for the contacting stepof the methods to further comprise contacting an in-dwelling device in apatient. In-dwelling devices include, but are not limited to, surgicalimplants, prosthetic devices and catheters, i.e., devices that areintroduced to the body of an individual and remain in position for anextended time. Such devices include, for example, artificial joints,heart valves, pacemakers, vascular grafts, vascular catheters,cerebrospinal fluid shunts, urinary catheters, and continuous ambulatoryperitoneal dialysis (CAPD) catheters.

A quinolone, particularly a gemifloxacin compound or composition of theinvention may be administered by injection to achieve a systemic effectagainst relevant bacteria, preferably a pneumococcal or Haemophiluspathogenic bacteria, shortly before insertion of an in-dwelling device.Treatment may be continued after surgery during the in-body time of thedevice. In addition, the composition could also be used to broadenperioperative cover for any surgical technique to prevent bacterialwound infections caused by or related to pneumococcal or Haemophiluspathogenic bacteria.

In addition to the therapy described above, a gemifloxacin compound orcomposition used in the methods of this invention may be used generallyas a wound treatment agent to prevent adhesion of bacteria to matrixproteins, particularly pneumococcal or Haemophilus pathogenic bacteria,exposed in wound tissue and for prophylactic use in dental treatment asan alternative to, or in conjunction with, antibiotic prophylaxis.

Alternatively, a quinolone, particularly a gemifloxacin compound orcomposition of the invention may be used to bathe an indwelling deviceimmediately before insertion. The active agent will preferably bepresent at a concentration of 1 μg/ml to 10 mg/ml for bathing of woundsor indwelling devices.

Also provided by the invention is a method of treating or preventing abacterial infection by pneumococcal or Haemophilus pathogenic bacteriacomprising the step of administering an antibacterially effective amountof a composition comprising a quinolone, particularly a gemifloxacincompound to a mammal, preferably a human, suspected of having or beingat risk of having an infection with pneumococcal or Haemophiluspathogenic bacteria.

A preferred object of the invention provides a method wherein saidpneumococcal pathogenic bacteria is selected from the group consistingof: bacteria comprising a mutation in a quinolone resistance-determiningregion (QRDR) of parc, gyrA, parE, and/or gyrB; bacteria comprising amutation in ParC at S79-F or Y, D83-N, R95-C, or K137-N; bacteriacomprising a mutation in gyrA at S83-A, C, F, or Y; E87-K; or S116-G;bacteria comprising a mutation in parE at D435-N or I460-V; bacteriacomprising a mutation in gyrB at D435-N or E474-K; bacteria comprisingat least four mutations in a QRDR or parC, gyrA, parE, and gyrB;bacteria comprising a mutation in a quinolone resistance-determiningregion (QRDR) of parC, gyrA, parE, and/or gyrB; bacteria that areciprofloxacin-resistant, levofloxacin-resistant, sparfloxacin-resistant,grepafloxacin-resistant, or trovafloxacin-resistant, or a combinationthereof, that comprise a mutation in ParC at S79-F or Y, D83-N, R95-C,or K137-N; bacteria that are ciprofloxacin-resistant,levofloxacin-resistant, sparfloxacin-resistant, grepafloxacin-resistant,or trovafloxacin-resistant, or a combination thereof, that comprise amutation in gyrA at S83-A, C, F, or Y; E87-K; or S116-G; bacteria thatare ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in parE at D435-N or I460-V; bacteria that areciprofloxacin-resistant, levofloxacin-resistant, sparfloxacin-resistant,grepafloxacin-resistant, or trovafloxacin-resistant, or a combinationthereof, that comprise a mutation in gyrB at D435-N or E474-K; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise atleast four mutations in a QRDR or parC, gyrA, parE, and gyrB; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in a quinolone resistance-determining region (QRDR) of parC,gyrA, parE, and/or gyrB; Streptococcus pneumoniae bacteria comprising amutation in ParC at 879-F or Y, D83-N, R95-C, or K137-N; Streptococcuspneumoniae bacteria comprising a mutation in gyrA at S83-A, C, F, or Y;E87-K; or S116-G; Streptococcus pneumoniae bacteria comprising amutation in parE at D435-N or I460-V; Streptococcus pneumoniae bacteriacomprising a mutation in gyrB at D435-N or E474-K; Streptococcuspneumoniae bacteria comprising at least four mutations in a QRDR orparC, gyrA, parE, and gyrB; and Streptococcus pneumoniae bacteriacomprising a mutation in a quinolone resistance-determining region(QRDR) of parC, gyrA, parE, and/or gyrB.

A preferred object of the invention provides a method wherein saidquinolone-resistant pneumococcal pathogenic bacteria is selected fromthe group consisting of: a pneumococcal strain comprising a mutation inthe quinolone resitance-determining region (QRDR) of parC and/or gyrA; apneumococcal strain comprising a mutation in parC said mutationcomprising S79-F and/or Y, D83-G and/or N, N91-D, R95-C, and/or K137-N;a pneumococcal strain comprising a mutation in gyrA said mutationcomprising S81-A, C, F, and/or Y; E85-K; and/or S114-G; a pneumococcalstrain comprising a mutation in parE said mutation comprising D435-Nand/or I460-V; a pneumococcal strain comprising a mutation in gyrB saidmutation comprising D435-N and/or E474-K; a pneumococcal straincomprising a mutation in comprising three or four mutations in a QRDRsof parC, gyrA, parE, and/or gyrB; a pneumococcal strain comprising amutation in comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB, any of which are resistant to ciprofloxacin,levofloxacin, or sparfloxacin; and a pneumococcal strain comprising amutation in comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB, any of which also comprising an efflux mechanism ofquinolone resistance.

A further preferred object of the invention provides a method whereinsaid rare pathogenic H. influenzae strain is selected from the groupconsisting of: bacteria comprising a mutation set forth in Table 11 or12; a Haemophilus influenzae strain set forth in Table 11 or 12;bacteria of the genus Haemophilhs comprising a mutation set forth inTable 11 or 12; and bacteria of the species Haemophilus influenzaecomprising a mutation set forth in Table 11 or 12.

Other pneumococcal and Haemophilus pathogenic bacteria may also beincluded in the methods. The skilled artisan may identify theseorganisms as provided herein as well as using other methods known in theart, e.g. MIC tests.

Preferred embodiments of the invention include, among other things,methods wherein said composition comprises gemifloxacin, or apharmaceutically acceptable derivative thereof.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. This exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail.

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Example 1

Bacteria

For agar dilution MICs, quinolone susceptible pneumococci comprised 64penicillin susceptible (MICs ≦0.06 μg/ml), 68 penicillin intermediate(MICs 0.125-1.0 μg/ml) and 75 penicillin resistant (MIC 2.0-16.0 μg/ml)strains (all quinolone susceptible, with ciprofloxacin MICs ≦4.0 μg/ml).All susceptible, and some intermediate and resistant strains, wererecent U.S. isolates. The remainder of intermediate and resistantstrains were isolated in South Africa, Spain, France, Central andEastern Europe, and Korea. Additionally, 28 strains with ciprofloxacinMICs ≧8 μg/ml some from a collection of organisms were tested by agardilution. Additionally these strains were tested for mutations in parC,gyrA, parE, and gyrB (Pan, et al., Antimicrob. Agents Chemother.40:2321-2326, 1996) and for efflux mechanism (Brenwald, et al.,Antimicrob. Agents Chemother. 42:2032-2035, 1998). For time-killstudies, 4 penicillin susceptible, 4 intermediate and 4 resistantstrains (2 quinolone resistant) were tested, while for PAE studies 5quinolone susceptible and 1 resistant strains were studied.

Example 2

Antimicrobials and MIC Testing

Agar dilution methodology was performed on 234 strains as describedpreviously (M. R. Jacobs, Clin. Infect. Dis. 15:119-127, 1992; Jacobs,et al., Rev. Med. Microbiol. 6:77-93, 1995), using Mueller-Histon agar(BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% sheepblood. Broth MICs for 12 strains tested by time-kill and 6 tested by PAEwere performed according to NCCLS recommendations (Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,3rd Edition, NCCLS, Villanova, Pa.) using cation-adjusted Mueller-Hintonbroth with 5% lysed defibrinated horse blood. Standard quality controlstrains, including Streptococcus pneumoniae ATCC 49619, were included ineach run of agar and broth dilution MICs.

Example 3

Time-kill Testing

For time-kill studies, glass tubes containing 5 ml cation-adjustedMueller-Hinton broth (Difco) +5% lysed horse blood with doublingantibiotic concentrations were inoculated with 5×10⁵ to 5×10⁶ cfu/ml andincubated at 35° C. in a shaking water bath. Antibiotic concentrationswere chosen to comprise 3 doubling dilutions above and 3 dilutions belowthe agar dilution MIC. Growth controls with inoculum but no antibioticwere included with each experiment (Pankuch, et al., Antimicrob. AgentsChemother. 38:2065-2072, 1994; Pankuch, et al., Antimicrob. AgentsChemother 40:1653-1656, 1996).

Lysed horse blood was prepared as described previously. The bacterialinoculum was prepared by diluting a 16 h broth (medium as above) culturein the same medium. Dilutions required to obtain the correct inoculum(5×10⁵-5×10⁶ cfu/ml) were determined by prior viability studies usingeach strain (Pankuch, et al., Antimicrob. Agents Chemother.38:2065-2072, 1994; Pankuch, et al., Antimicrob. Agents Chemother.40:1653-1656, 1996).

To inoculate each tube of serially diluted antibiotic, 50 ìof dilutedinoculum was delivered by pipette beneath the surface of the broth.Tubes were then vortexed and plated for viability counts within 10 min(approximately 0.2 h). The original inoculum was determined by using theuntreated growth control. Only tubes containing an initial inoculumwithin the range of 5×10⁵ to 5×10⁶ cfu/ml were acceptable (Pankuch, etal., Antimicrob. Agents Chemother. 38:2065-2072, 1994; Pankuch, et al.,Antimicrob. Agents Chemother. 40:1653-1656, 1996).

Viability counts of antibiotic-containing suspensions were performed byplating ten-fold dilutions of 0.1 ml aliquots from each tube in sterileMueller-Hinton broth onto trypticase soy agar 5% sheep blood agar plates(BBL). Recovery plates were incubated for up to 72 h. Colony counts wereperformed on plates yielding 30-300 colonies. The lower limit ofsensitivity of colony counts was 300 cfu/ml (Pankuch, et al.,Antimicrob. Agents Chemother. 38:2065-2072, 1994; Pankuch, et al.,Antimicrob. Agents Chemother. 40:1653-1656, 1996).

Time-kill assays were analysed by determining the number of strainswhich yielded a log₁₀ cfu/ml of −1, −2 and −3 at 0, 3, 6, 12 and 24 h,compared to counts at time 0 h. Antimicrobials were consideredbactericidal at the lowest concentration that reduced the originalinoculum by ≧3 log₁₀ cfu/ml (99.9%) at each of the time periods, andbacteriostatic if the inoculum was reduced by 0-3 log₁₀ cfu/ml. With thesensitivity threshold and inocula used in these studies, no problemswere encountered in delineating 99.9% killing, when present. The problemof bacterial carryover was addressed as described previously. Formacrolide time-kill testing, only strains with MICs ≦4.0 μg/ml weretested (Pankuch, et al., Antimicrob. Agents Chemother. 38:2065-2072,1994; Pankuch, et al., Antimicrob. Agents Chemother. 40:1653-1656,1996).

Example 4

Post-antibiotic Effect Testing

The post-antibiotic effect (PAE) (Craig, et al., V. Lorian (ed.)Antibiotics in Laboratory Medicine, Williams and Wilkins, Baltimore,pages 296-329, 1996) was determined by the viable plate count method,using Mueller-Hinton broth (MHB) supplemented with 5% lysed horse bloodwhen testing pneumococci. The PAE was induced by exposure to 10×MIC for1 h (Craig, et al., V. Lorian (ed.) Antibiotics in Laboratory Medicine,Williams and Wilkins, Baltimore, pages 296-329, 1996; Spangler, et al.,Antimicrob. Agents Chemother. 41:2173-2176, 1997; Spangler, et al.,Antimicrob. Agents Chemother. 42:1253-1255, 1998.) Additionally, the onequinolone resistant strain was exposed at quinolone concentrations5×MIC. Tubes containing 5 ml broth with antibiotic were inoculated withapproximately 5×10⁶ cfu/ml. Growth controls with inoculum but noantibiotic were included with each experiment. Tubes were placed in ashaking water bath at 35° C. for 1 h. At the end of the exposure period,cultures were diluted 1:1000 to remove antibiotic. A control containingbacteria pre-exposed to antibiotic at a concentration of 0.01×MIC wasalso prepared (Spangler, et al., Antimicrob. Agents Chemother.41:2173-2176, 1997; Spangler, et al., Antimicrob. Agents Chemother.42:1253-1255, 1998).

Viability counts were determined before exposure and immediately afterdilution (0 h), and then every 2 h until tube turbidity reached a #1McFarland standard. Inocula were prepared by suspending growth from anovernight blood agar plate in broth. The broth was incubated at 35° C.for 2-4 h in a shaking water bath until turbidity matched a #1 McFarlandstandard, and checked for viability by plate counts (Spangler, et al.,Antimicrob. Agents Chemother. 41:2173-2176, 1997; Spangler, et al.,Antimicrob. Agents Chemother. 42:1253-1255, 1998).

The PAE was defined as PAE=T-C; T=time required for viability counts ofan antibiotic-exposed culture to increase by 1 log₁₀ above countsimmediately after dilution; C=corresponding time for growth control. Foreach experiment, viability counts (log₁₀ cfu/ml) were plotted againsttime, and results expressed as the mean of two separate assays ±SD(Craig, et al., V. Lorian (ed.), Antibiotics in Laboratory Medicine,Williams and Wilkins, Baltimore, pages 296-329, 1996).

Example 5

PCR of Quinolone Resistance Determinants and DNA Sequence Analysis

Polymerase chain reaction method (PCR) was used to amplify parC, parE,gyrA, and gyrB using primers and cycling conditions described by Pan andFisher (Pan, et al., Antimicrob. Agents Chemother. 40:2321-2326, 1996).Template DNA for PCR was prepared using Prep-A-Gene kit (Bio-Rad,Hercules, Calif.) as recommended by the manufacturer. Afteramplification PCR products were purified from excess primers andnucleotides using QIAquick PCR Purification kit as recommended by themanufacturer (Qiagen, Valencia, Calif.) and sequenced directly usingApplied Biosystems Model 373A DNA sequencer. Strains with mutationswidely described in the literature (e.g. Ser79-Tyr or Phe in ParC andSer83-Tyr or Phe in GyrA) were sequenced once in the forward direction.Strains with no mutations in any of the above mentioned genes or with apreviously undescribed mutation were sequenced twice in the forwarddirection and once in the reverse direction on products of independentPCR reactions (Davies, et al., Antimicrob. Agents Chemother.43:1177-1182, 1999).

Example 6

Determination of Efflux Mechanism

MICs were determined in the presence and absence of 10 μg/ml ofreserpine (Sigma Chemicals, St. Louis, Mo.) as known in the art. Strainswith at least a twofold lower ciprofloxacin MIC in the presence ofreserpine were then tested against the other quinolones in the presenceof reserpine. Results were repeated three times Brenwald, et al.,Antimicrob. Agents Chemother. 42:2032-2035, 1998; Davies, et al.,Antimicrob. Agents Chemother. 43:1177-1182, 1999).

Example 7

Bacterial Strains

28 strains with ciprofloxacin MICs ≧8 μg/ml were tested by agardilution. Additionally these strains were tested for mutations in parC,gyrA, parE, and gyrB (Pan, et al., Antimicrob. Agents Chemother.40:2321-2326, 1996) and for efflux mechanism (Brenwald, et al.,Antimicrob. Agents Chemother. 42:2032-2035,1998).

Example 8

Antimicrobials and MIC Testing

Gemifloxacin susceptibility powder was obtained from SmithKline BeechamLaboratories, Harlow, UK. Agar dilution methodology was performed on 28strains as described previously (M. R. Jacobs, Clin. Infect. Dis.15:119-127, 1992 and M. R. Jacobs, Rev. Med. Microbiol. 6:77-93, 1995),using Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, Md.)supplemented with 5% sheep blood. Standard quality control strains,including Streptococcus pneumoniae ATCC 49619, were included in each runof agar dilution MICs.

Example 9

PCR of Quinolone Resistance Determinants and DNA Sequence Analysis

PCR was used to amplify parC, parE, gyrA and gyrB using primers andcycling conditions described by Pan et al (Pan et al., Antimicrob.Agents Chemother. 40:2321-2326, 1996). Template DNA for PCR was preparedusing Prep-A-Gene kit (Bio-Rad, Hercules, Calif., USA) as recommended bythe manufacturer. After amplification PCR products were purified fromexcess primers and nucleotides using QlAquick PCR Purification kit asrecommended by the manufacturer (Qiagen, Valencia, Calif., USA) andsequenced directly using Applied Biosystems Model 373A DNA sequencer.

Example 10

Determination of Efflux Mechanism

MICs were determined in the presence and absence of 10 μg/ml ofreserpine (Sigma row Chemicals, St. Louis, Mo., USA) as describedpreviously (Brenwald, et al., Antimicrob. Agents Chemother.42:2032-2035, 1998 and Davies, et al., Antimicrob. Agents Chemother.43:1177-1182, 1999). Strains with at least a twofold lower ciprofloxacinMIC in the presence of reserpine were then tested against the otherquinolones in the presence of reserpine. Results were repeated threetimes previously (Brenwald, et al., Antimicrob. Agents Chemother.42:2032-2035, 1998 and Davies, et al., Antimicrob. Agents Chemother.43:117714-1182, 1999).

Example 11

Bacterial Strains and Antimicrobials

Gemifloxacin susceptibility powder was obtained from SmithKline BeechamLaboratories, Harlow, UK.

Example 12

MIC Determination

Inocula were prepared from chocolate agar plates incubated for a full 24hours by the direct colony suspension method as follows: In a tube ofMueller-Hinton broth (Difco), an organism suspension was made to adensity of a 0.5 McFarland standard (1×10⁸ CFU/ml). The latter inoculumwas diluted in sterile saline such that final organisms suspensions intrays yielded colony counts of 3-8×10⁵ CFU/ml. Frozen microdilutiontrays were obtained from MicroMedia Systems, Inc. (Cleveland, Ohio,USA). Each tray contained all antimicrobials prepared in freshly madeHTM. Wells were inoculated with 100 μl suspensions and incubated inambient air at 35° C. for 20-24 hours. The lowest drug concentrationshowing no growth was read as the MIC. Standard quality control strains,including H. influenzae ATCC 49766, H. influenzae ATCC 49247,Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 wereincluded with each run.

Example 13

PCR and DNA Sequencing of Quinolone-resistant Determining Region ofparC, parE, gyrA, and gyrB

Template DNA for PCR was prepared as follows: a colony from ovemightgrowth was lysed by incubation for 1 hour at 37° C. in lysis buffer (6mM Tris-HCL [pH 7.4], 1 M NaCl, 10 mM EDTA [pH 8.0], 0.2% deoxycholate,0.5% sodium lauroyl sarcosine) to which lysozyme (Sigma, St. Louis, Mo.,USA) at 0.5 mg/ml and lysostaphin (Sigma) at 0.05 mg/ml were addedfresh. DNA was isolated from the lysed cells using a Prep-A-Gene kit(Bio-Rad, Hercules, Calif., USA) as recommended by the manufacturer. PCRwas carried out in a final volume of 100 μl containing 10 mM Tris-HCl(pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each dNTPS, 5 pmol of eachprimer, 5-10 ng DNA template, and 2.5 U Taq DNA polymerase (FisherBiotech). Conditions for PCR were 30 cycles of 94° C. for 1 minute,annealing at 53° C. for 1 minute, and extension at 72° C. from 3minutes. For parC a 370 bp region encoding residues 41 to 163 wasamplified using primers HFPARCUP (5′-TGGTTTAAAACCCGTTCA-3′, nucleotidepositions 120 to 137) (SEQ. ID NO:1), and HFPARCDN(5′-AGCAGGTAAATAITGTGG-3′, positions 473-490) (SEQ ID NO:2). For parE a471 bp region encoding residues 335 to 491 was amplified using primersHFPAREUP (5′-GAACGCTTATCATCACGCCA-3′, positions 1003 to 1022) (SEQ IDNO:3) and HFPAREDN (5′-AGCATCCGCGAGAATACAGA-3′, positions 1454 to 1473)(SEQ ID NO:4). For gyrA a 375 bp region encoding residues 47 to 171 wasamplified using primers BFGYRAUP (5′-CCGCCGCGTACTGTTCT-3′, positions 138to 154) (SEQ ID NO:5) and HFGYRADN (5′-CCATTTGCTAAAAGTGC-3′, positions496 to 512)(SEQ ID NO:6). For gyrB a 445 bp region encoding residues 367to 513 was amplified using primers HFGYRBFOR (5′-GGAAAATCCTGCAGATGC-3′,positions 1095 to 1113) (SEQ ID NO:7) and HFGYRBBAC(5′-AAGCAACGTACGGATGTG-3′, positions 1522 to 1539) (SEQ ID NO:8). Afteramplification PCR products were purified from excess primers andnucleotides using a QIAquick PCR purification kit (Qiagen, Valencia,Calif., USA) and sequenced directly by using an Applied Biosystems model373A DNA sequencer. All genes were sequenced twice in the forward andreverse directions on products of independent PCRs.

Each reference cited herein is hereby incorporated by reference in itsentirety. Moreover, each patent application to which this applicationclaims priority is hereby incorporated by reference in its entirety.

8 1 18 DNA Haemophilus influenzae 1 tggtttaaaa cccgttca 18 2 18 DNAHaemophilus influenzae 2 agcaggtaaa tattgtgg 18 3 20 DNA Haemophilusinfluenzae 3 gaacgcttat catcacgcca 20 4 20 DNA Haemophilus influenzae 4agcatccgcg agaatacaga 20 5 17 DNA Haemophilus influenzae 5 ccgccgcgtactgttct 17 6 17 DNA Haemophilus influenzae 6 ccatttgcta aaagtgc 17 7 18DNA Haemophilus influenzae 7 ggaaaatcct gcagatgc 18 8 18 DNA Haemophilusinfluenzae 8 aagcaacgta cggatgtg 18

What is claimed is:
 1. A method for modulating metabolism ofpneumococcal pathogenic bacteria comprising the step of contactingpneumococcal pathogenic bacteria with an antibacterially effectiveamount of a composition comprising a gemifloxacin compound, or anantibacterially effective derivative thereof, wherein said pneumococcalpathogenic bacteria is selected from the group consisting of: bacteriacomprising a mutation in a quinolone resistance-determining region(QRDR) of parC, gyrA, parE, and/or gyrB; bacteria comprising a mutationin parC at S79-F or Y, D83-N, R95-C, or K137-N; bacteria comprising amutation in gyrA at S83-A, C, F, or Y; E87-K; or S116-G; bacteriacomprising a mutation in parE at D435-N or I460-V; bacteria comprising amutation in gyrB at D435-N or E474-K; bacteria comprising at least fourmutations in a QRDR of parC, gyrA, parE, and gyrB; bacteria that areciprofloxacin-resistant, levofloxacin-resistant, sparfloxacin-resistant,grepafloxacin-resistant, or trovafloxacin-resistant, or a combinationthereof, that comprise a mutation in parC at S79-F or Y, D83-N, R95-C,or K137-N; bacteria that are ciprofloxacin-resistant,levofloxacin-resistant, sparfloxacin-resistant, grepafloxacin-resistant,or trovafloxacin-resistant, or a combination thereof, that comprise amutation in gyrA at S83-A, C, F, or Y; E87-K; or S116-G; bacteria thatare ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in parE at D435-N or I460-V; bacteria that areciprofloxacin-resistant, levofloxacin-resistant, sparfloxacin-resistant,grepafloxacin-resistant, or trovafloxacin-resistant, or a combinationthereof, that comprise a mutation in gyrB at D435-N or E474-K; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise atleast four mutations in a QRDR of parC, gyrA, parE, and gyrB; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in a quinolone resistance-determining region (QRDR) of parC,gyrA, parE, and/or gyrB; Streptococcus pneumoniae bacteria comprising amutation in parC at S79-F or Y, D83-N, R95-C, or K137-N; Streptococcuspneumoniae bacteria comprising a mutation in gyrA at S83-A, C, F, or Y;E87-K; or S116-G; Streptococcus pneumoniae bacteria comprising amutation in parE at D435-N or I460-V; Streptococcus pneumoniae bacteriacomprising a mutation in gyrB at D435-N or E474-K; Streptococcuspneumoniae bacteria comprising at least four mutations in a QRDR ofparC, gyrA, parE, and gyrB; and Streptococcus pneumoniae bacteriacomprising a mutation in a quinolone resistance-determining region(QRDR) of parc, gyrA, parE, and/or gyrB.
 2. The method of claim 1wherein said modulating metabolism is inhibiting growth of saidbacteria.
 3. The method of claim 1 wherein said modulating metabolism iskilling said bacteria.
 4. The method of claim 1 wherein said contactingsaid bacteria comprises the further step of introducing said compositioninto a mammal.
 5. The method of claim 4 wherein said mammal is a human.6. A method of treating or preventing a bacterial infection bypneumococcal pathogenic bacteria comprising the step of administering anantibacterially effective amount of a composition comprising agemifloxacin compound, or an antibacterially effective derivativethereof, to a mammal suspected of having or being at risk of having aninfection with pneumococcal pathogenic bacteria, wherein saidpneumococcal pathogenic bacteria is selected from the group consistingof: bacteria comprising a mutation in a quinolone resistance-determiningregion (QRDR) of parC, gyrA, parE, and/or gyrB; bacteria comprising amutation in parC at S79-F or Y, D83-N, R95-C, or K137-N; bacteriacomprising a mutation in gyra at S83-A, C, F, or Y; E87-K; or S116-G;bacteria comprising a mutation in parE at D435-N or I460-V; bacteriacomprising a mutation in gyrB at D435-N or E474-K; bacteria comprisingat least four mutations in a QRDR of parC, gyrA, parE, and gyrB;bacteria that are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in parC at S79-F or Y, D83-N, R95-C, or K137-N; bacteria thatare ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in gyrA at S83-A, C, F, or Y; E87-K; or S116-G; bacteria thatare ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in parE at D435-N or I460-V; bacteria that areciprofloxacin-resistant, levofloxacin-resistant, sparfloxacin-resistant,grepafloxacin-resistant, or trovafloxacin-resistant, or a combinationthereof, that comprise a mutation in gyrB at D435-N or E474-K; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfloxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise atleast four mutations in a QRDR of parC, gyrA, parE, and gyrB; bacteriathat are ciprofloxacin-resistant, levofloxacin-resistant,sparfioxacin-resistant, grepafloxacin-resistant, ortrovafloxacin-resistant, or a combination thereof, that comprise amutation in a quinolone resistance-determining region (QRDR) of parC,gyrA, parE, and/or gyrB; Streptococcus pneumoniae bacteria comprising amutation in parC at S79-F or Y, D83-N, R95-C, or K137-N; Streptococcuspneumoniae bacteria comprising a mutation in gyrA at S83-A, C, F, or Y;E87-K; or S116-G; Streptococcus pneumoniae bacteria comprising amutation in parE at D435-N or I460-V; Streptococcus pneumoniae bacteriacomprising a mutation in gyrB at D435-N or E474-K; Streptococcuspneumoniae bacteria comprising at least four mutations in a QRDR ofparC, gyrA, parE, and gyrB; and Streptococcus pneumoniae bacteriacomprising a mutation in a quinolone resistance-determining region(QRDR) of parC, gyrA, parE, and/or gyrB.
 7. The method of claim 6wherein said mammal is a human.
 8. The method of claim 6 wherein saidpneumococcal pathogenic bacteria is a Streptococcus pneumoniae bacteriacomprising a mutation in parC at S79-F or Y, D83-N, R95-C, or K137-N. 9.The method of claim 6 wherein said pneumococcal pathogenic bacteria is aStreptococcus pneumoniae bacteria comprising a mutation in gyrA atS83-A, C, F, or Y; E87-K; or S116-G.
 10. The method of claim 6 whereinsaid pneumococcal pathogenic bacteria is a Streptococcus pneumoniaebacteria comprising a mutation in parE at D435-N or I460-V.
 11. Themethod of claim 6 wherein said pneumococcal pathogenic bacteria is aStreptococcus pneumoniae bacteria comprising a mutation in gyrB atD435-N or E474-K.
 12. The method of claim 6 wherein said pneumococcalpathogenic bacteria is a Streptococcus pneumoniae bacteria comprising atleast four mutations in a QRDR of parC, gyrA, parE, and gyrB.
 13. Themethod of claim 6 wherein said pneumococcal pathogenic bacteria is aStreptococcus pneumoniae bacteria comprising a mutation in a quinoloneresistance-determining region (QRDR) of parC, gyra, parE, and/or gyrB.14. A method for modulating metabolism of quinolone-resistantpneumococcal pathogenic bacteria comprising the step of contactingquinolone-resistant pneumococcal pathogenic bacteria with anantibacterially effective amount of a composition comprising agemifloxacin compound, or an antibacterially effective derivativethereof, wherein said quinolone-resistant pneumococcal pathogenicbacteria is selected from the group consisting of: a pneumococcal straincomprising a mutation in the quinolone resistance-determining region(QRDR) of parC and/or gyrA; a pneumococcal strain comprising a mutationin parC, said mutation comprising S79-F and/or Y, D83-G and/or N, N91-D,R95-C, and/or K137-N; a pneumococcal strain comprising a mutation ingyrA, said mutation comprising S81-A, C, F, and/or Y; E85-K; and/orS114-G; a pneumococcal strain comprising a mutation in parE, saidmutation comprising D435-N and/or I460-V; a pneumococcal straincomprising a mutation in gyrB, said mutation comprising D435-N and/orE474-K; a pneumococcal strain comprising three or four mutations in aQRDRs of parC, gyrA, parE, and/or gyrB; a pneumococcal strain comprisingthree or four mutations in a QRDRs of parC, gyrA, parE, and/or gyrB, anyof which are resistant to ciprofloxacin, levofloxacin, or sparfloxacin;and a pneumococcal strain comprising three or four mutations in a QRDRsof parC, gyrA, parE, and/or gyrB, any of which also comprising an effluxmechanism of quinolone resistance.
 15. The method of claim 14 whereinsaid modulating metabolism is inhibiting growth of said bacteria. 16.The method of claim 14 wherein said modulating metabolism is killingsaid bacteria.
 17. The method of claim 14 wherein said contacting saidbacteria comprises the further step of introducing said composition intoa mammal.
 18. The method of claim 17 wherein said mammal is a human. 19.The method of claim 14 wherein said quinolone-resistant pneumococcalpathogenic bacteria is a pneumococcal strain comprising a mutation inparC, said mutation comprising S79-F and/or Y, D83-G and/or N, N91-D,R95-C, and/or K137-N.
 20. The method of claim 14 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising a mutation in gyrA, said mutation comprising S81-A, C,F, and/or Y; E85-K; and/or S114-G.
 21. The method of claim 14 whereinsaid quinolone-resistant pneumococcal pathogenic bacteria is apneumococcal strain comprising a mutation in parE, said mutationcomprising D435-N and/or I460-V.
 22. The method of claim 14 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising a mutation in gyrB, said mutation comprising D435-Nand/or E474-K.
 23. The method of claim 14 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB.
 24. The method of claim 14 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB, any of which are resistant to ciprofloxacin,levofloxacin, or sparfloxacin.
 25. The method of claim 14 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB, any of which also comprising an efflux mechanism ofquinolone resistance.
 26. A method of treating or preventing a bacterialinfection by quinolone-resistant pneumococcal pathogenic bacteriacomprising the step of administering an antibacterially effective amountof a composition comprising a gemifloxacin compound, or anantibacterially effective derivative thereof, to a mammal suspected ofhaving or being at risk of having an infection with quinolone-resistantpneumococcal pathogenic bacteria, wherein said quinolone-resistantpneumococcal pathogenic bacteria is selected from the group consistingof: a pneumococcal strain comprising a mutation in the quinoloneresistance-determining region (QRDR) of parC and/or gyrA; a pneumococcalstrain comprising a mutation in parC, said mutation comprising S79-Fand/or Y, D83-G and/or N, N91 -D, R95-C, and/or K137-N; a pneumococcalstrain comprising a mutation in gyrA, said mutation comprising S81-A, C,F, and/or Y; E85-K; and/or S114-G; a pneumococcal strain comprising amutation in parE, said mutation comprising D435-N and/or I460-V; apneumococcal strain comprising a mutation in gyrB, said mutationcomprising D435-N and/or E474-K; a pneumococcal strain comprising threeor four mutations in a QRDRs of parC, gyrA, parE, and/or gyrB; apneumococcal strain comprising three or four mutations in a QRDRs ofparC, gyrA, parE, and/or gyrB, any of which are resistant tociprofloxacin, levofloxacin, or sparfloxacin; and a pneumococcal straincomprising three or four mutations in a QRDRs of parC, gyrA, parE,and/or gyrB, any of which also comprising an efflux mechanism ofquinolone resistance.
 27. The method of claim 14 wherein said mammal isa human.
 28. The method of claim 26 wherein said quinolone-resistantpneumococcal pathogenic bacteria is a pneumococcal strain comprising amutation in parC, said mutation comprising S79-F and/or Y, D83-G and/orN, N91-D, R95-C, and/or K137-N.
 29. The method of claim 26 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising a mutation in gyrA, said mutation comprising S81-A, C,F, and/or Y; E85-K; and/or S114-G.
 30. The method of claim 26 whereinsaid quinolone-resistant pneumococcal pathogenic bacteria is apneumococcal strain comprising a mutation in parE, said mutationcomprising D435-N and/or I460-V.
 31. The method of claim 26 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising a mutation in gyrB, said mutation comprising D435-Nand/or E474-K.
 32. The method of claim 26 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB.
 33. The method of claim 26 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB, any of which are resistant to ciprofloxacin,levofloxacin, or sparfloxacin.
 34. The method of claim 26 wherein saidquinolone-resistant pneumococcal pathogenic bacteria is a pneumococcalstrain comprising three or four mutations in a QRDRs of parC, gyrA,parE, and/or gyrB, any of which also comprising an efflux mechanism ofquinolone resistance.